The present invention relates to a method for manufacturing organic acid by high-efficiency fermentation, more specifically, to a method for manufacturing organic acid in a highly efficient manner by culturing organic acid bacteria in a cell-recycle multiple-stage continuous fermentor.
Organic acids such as lactic acid, acetic acid, and citric acid which are widely used in industry have been manufactured by chemical synthesis, enzyme reaction or microbial fermentation process. However the cost-efficiency problem has limited the availability of manufacturing processes other than chemical synthesis, and lots of efforts have been made to replace the chemical process with more environment-friendly fermentation process. The importance of new process has been stressed by the discovery of microorganisms capable of producing organic acids, the development of high-efficiency fermentation process, and the spreading of the cognition about environmental problems. Although the fermentation technology is usually employed in the manufacturing process for acetic acid and citric acid, relatively low yield for other organic acids has limited, or sometimes banned, the practical use of fermentation process (see: Roehr, M., Products of Primary Metabolism, in: Biotechnology, 2nd Ed., Vol. 6(Rehm, H,-J., Reed, G., Eds.), Weinheim: Verlag Chemie, 1996).
Naturally, a variety of approaches have been made to establish a fermentation process for a very important organic acid of lactic acid whose physical properties and fermentation conditions have been known, and to connect the result of lactic acid production fermentation with those of other organic acids. In line with these activities, lactic acid fermentation using naturally abundant materials such as starch, glucose, sugar and lactose has consistently increased and high-efficiency fermentation process has been sought to meet the enormous potential need, while the chemical synthesis of lactic acid has gradually decreased.
Lactic acids which are produced in the metabolic pathway of living organisms, are contained in numerous fermented foods such as Kimchi (a Korean traditional fermented product) and Yakult. The lactic acids have been manufactured mainly in U.S.A. and European contries since its first identification by Scheele in 1780. Lactic acid which is optically active, due to a chiral carbon, is classified as L- and D-lactic acids, and humans are able to utilize only L-form. Lactic acids are produced by microbial fermentation in a form of L- or D-lactic acid, sometimes racemic mixture of the two forms, depending on the specificity of enzymes of the microorganisms.
Though the world market of lactic acid in the fields of foodstuff and cosmetics is somewhat limited, new demands in the biodegradable polymers and diverse solvents have rapidly grown. To meet the needs in the art, it is essentially required to manufacture lactic acid by a novel, high-efficiency method, compared to the conventional processes. Lactic acid-based biopolymer can be degraded easily under the environmental condition, and has good properties in light of mechanical stability, which makes plausible the use of lactic acid as a base material for polymer synthesis.
Until now, lactic acid has been manufactured by employing the batch-type fermentation process which comprises the steps of culturing lactic acid bacteria for more than 60 hours after inoculation and isolating lactic acid from the culture medium. The batch process has an advantage of high-concentration lactic acid production of over 120 g/L, though it has revealed a critical shortcoming of low volumetric productivity of less than 5 g/L/h. To compensate for the low productivity, the fermentation volume is enlarged, which in turn increases the cost for the construction of fermentation facilities and the maintenance of the facilities.
As an alternative approach to overcome the low productivity of batch-type fermentation process, cell-recycling method by which microorganisms are concentrated in a fermentor to increase the productivity, has been suggested in the art. This method employs the principle that the concentration of the enzyme for lactic acid synthesis increases as the cell density in the fermentor does, which comprises the steps of: maintaining lactic acid bacteria in a fermentor by using isolation techniques such as centrifugation and membrane separation; culturing the lactic acid bacteria in a medium containing sugar as a major component; and, collecting the culture containing lactic acid.
Cell-recycling method using membrane separation has been studied in the U.S.A. since 1980""s, and in 1987, Cheryan et al reported that the volumetric lactic acid productivity of 84 g/L/h and the lactic acid concentration of 117 g/L can be realized (see: Mehaia, M. A. and M. Cheryan, Process Biochemistry, December 185-188, 1987). Cheryan et al also successfully obtained the volumetric lactic acid productivity of 22 g/L/h and the lactic acid concentration of 89 g/L (see: Tejayadi, S. and M. Cheryan, Appl. Microbiol. Biotechnol., 43:242-248, 1995). However, the prior art method is proven to be less satisfactory in a sense that the said results is not reproducible under the normal condition for continuous fermentation (see: Timmer, J. M. and J. Kromkamp, FEMS Microbiology Reviews, 14:29-38, 1994). Therefore, the productivity of 22 g/L/h and the concentration of 89 g/L of lactic acid ferment has been known to be the highest efficiency and concentration among the Cheryan""s results in 1995.
Although many results have been published using cell-recycling method in the continuous-type fermentation of lactic acid, Cheryan et al""s result is distinguished from the others in light of the high concentration of over 90 g/L of lactic acid ferment. The difficulty in obtaining high concentration of lactic acid is caused by the severe end-product inhibition, which is brought about at the lactic acid concentration of over 50 g/L.
On the other hand, multiple-stage continuous fermentation has been developed to attenuate the end-product inhibition in the course of lactic acid fermentation, in which the steps of culturing microorganisms and producing lactic acid are separated to give a high productivity: That is, using a multiple-stage continuous fermentor with two or more fermentors serially connected with each other, bacterial growth was facilitated in the first fermentor containing a relatively low concentration of target product to maintain the lactic acid production rate of the microorganism, and the target product was manufactured in the subsequent fermentors to reach the desired high concentration.
Actually, the multiple-stage continuous fermentation comprising two or 10 stages has been reported to allow the high-efficiency production of ethanol, a typical product showing the end-product inhibition, and it could be applied in other cases such as lactic acid, monoclonal antibody, enzyme and liquor as well (see: Gooijer, C. D., et al, Enzyme-Micorb. Technol., 18:202-219, 1996). Based on the perception about the end-product inhibition of lactic acid, which shows the same phenomenon as in ethanol, Mulligan et al performed two- and three-stages of continuous fermentation and improved the productivity of lactic acid by 25% and 57%, compared to single-stage method, respectively. However, the productivity of Mulligan et al""s was relatively low (e.g., 3xcx9c5 g/L/h), due to low cell concentration of 2xcx9c3 g/L (see: Mulligan, C. N., and B. F. Safi, Biotechnol. Bioeng., 38:1173, 1991).
Under these circumstances, many attempts have been made to produce lactic acid with a high-efficiency, by combining the cell-recycling method and the multiple-stage continuous fermentation method. However, it has revealed a problem that the production efficiency is not improved any more, compared to the chemical synthesis and batch-type fermentation. Accordingly, methods for manufacturing lactic acid with a high concentration and efficiency have been actively studied in the art, since the high efficiency production of lactic acid can be applied to the other organic acids such as acetic acid, formic acid, citric acid, malic acid, maleic acid, fumaric acid and succinic acid.
Nabisco Brands Inc. used serially connected two-stage fermentors to increase the final concentration of acetic acid and membranes between the said two fermentors in order to concentrate microorganisms in each fermentor (see: U.S. Pat. No. 4,456,622). However, it turned out to be a single fermentor in which two batch-type fermentors are connected with each other simply to reuse the microorganisms after fermentation. Accordingly, acetic acid cannot be produced with a high concentration and productivity, compared to the chemical synthesis.
Therefore, needs have continued to exist for the development of a novel method for manufacturing lactic acid in a highly efficient manner, which can be applied to the production of other organic acids.
The present inventors have tried to establish an improved method for manufacturing highly concentrated lactic acid by high-efficiency fermentation by employing a cell-recycle multiple-stage continuous fermentor with two-stage serially connected fermentors, and found that lactic acid can be manufactured with a high concentration of over 90 g/L and productivity of over 50 g/L/h, which can be effectively applied to the other organic acid fermentation processes showing the end-product inhibition.
A primary object of the present invention is, therefore, to provide a method for manufacturing organic acid with a high concentration and productivity.
The other object of the invention is to provide a cell-recycle multiple-stage continuous fermentor with serially connected fermentors.